Extracellular vesicles secreted by human aneuploid embryos present a distinct transcriptomic profile and upregulate MUC1 transcription in decidualised endometrial stromal cells.

STUDY QUESTION: Do extracellular vesicles (EVs) secreted by aneuploid human embryos possess a unique transcriptomic profile that elicits a relevant transcriptomic response in decidualized primary endometrial stromal cells (dESCs)? SUMMARY ANSWER: Aneuploid embryo-derived EVs contain transcripts of PPM1J, LINC00561, ANKRD34C, and TMED10 with differential abundance from euploid embryo-derived EVs and induce upregulation of MUC1 transcript in dESCs. WHAT IS KNOWN ALREADY: We have previously reported that IVF embryos secrete EVs that can be internalized by ESCs, conceptualizing that successful implantation to the endometrium is facilitated by EVs. Whether these EVs may additionally serve as biomarkers of ploidy status is unknown. STUDY DESIGN SIZE DURATION: Embryos destined for biopsy for preimplantation genetic testing for aneuploidy (PGT-A) were grown under standard conditions. Spent media (30 µl) were collected from euploid (n = 175) and aneuploid (n = 140) embryos at cleavage (Days 1-3) stage and from euploid (n = 187) and aneuploid (n = 142) embryos at blastocyst (Days 3-5) stage. Media samples from n = 35 cleavage-stage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocysts were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. PARTICIPANTS/MATERIALS SETTING METHODS: EVs were isolated from pools of media by differential centrifugation and EV-RNA sequencing was performed following a single-cell approach that circumvents RNA extraction. ESCs were decidualized (estradiol: 10 nM, progesterone: 1 µM, cAMP: 0.5 mM twice every 48 h) and incubated for 24 h with EVs (50 ng/ml). RNA sequencing was performed on ESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc = -5.13, P = 0.011), LINC00561 (log2fc = -7.87, P = 0.010), and ANKRD34C (log2fc = -7.30, P = 0.017) and more abundant in TMED10 (log2fc = 1.63, P = 0.025) compared to EVs of euploid embryos. Decidualization per se induced downregulation of MUC1 (log2fc = -0.54, P = 0.0028) in ESCs as a prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualized ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2fc = 0.85, P = 0.0201). LARGE SCALE DATA: Raw data have been uploaded to GEO (accession number GSE234338). LIMITATIONS REASONS FOR CAUTION: The findings of the study will require validation utilizing a second cohort of EV samples. WIDER IMPLICATIONS OF THE FINDINGS: The discovery that the transcriptomic profile of EVs secreted from aneuploid cleavage stage embryos differs from that of euploid embryos supports the possibility to develop a non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. STUDY FUNDING/COMPETING INTERESTS: The study was supported by a Marie Sklodowska-Curie Actions fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd. The authors have no conflicts of interest to declare.

The relationship between CYP19A1 gene expression in luteinized granulosa cells and follicular estradiol output in women with endometriosis.

Endometriosis was claimed to negatively affect the intrafollicular environment, hindering oocyte competence. Previous studies evaluated expression levels of cytochrome P450 aromatase (CYP19A) in granulosa and cumulus oophorus cells collected from endometriosis women, but results are controversial. To further investigate the intrafollicular environment whose alteration may potentially disturb ovarian steroidogenesis in endometriosis, gene expression of CYP19A and of its upstream enzymes, StAR and 3ßHSD was assessed in luteinized granulosa cells isolated from follicular fluids (FF) collected during Assisted Reproduction Technology (ART) procedures in women with stage III-IV disease and from subjects without the condition. In a subgroup of patients, cumulus oophorus cells (COCs) were also assessed for CYP19A, StAR and 3ßHSD gene expression. No difference in mRNA expression of CYP19A1, StAR and 3ßHSD in both granulosa cells and COCs was observed between the two groups of patients. No significant difference was also found between estradiol FF levels detected in endometriosis patients (median=873, IQR=522-1221 ng/ml)) and control patients (median=878, IQR=609-1137 ng/ml). To gain more insight into the intrafollicular regulation of CYP19A in patients with endometriosis, associations between expression of the analyzed genes, systemic and follicular 17ß-estradiol levels and ART outcomes were assessed. While in the control group, levels of CYP19A1, StAR and 3ßHSD transcripts significantly correlated with follicular estradiol levels (adjusted R² of 0.60), no significant association was detected in affected women (adjusted R² of 0.23). After stratification of the populations based on the presence of the disease, CYP19A1 expression was shown to correlate with the number of oocytes retrieved [ß:- 1.214;95%CI: - 2.085 - (-0.343); p = 0.007] in the control group while this association was not present in patients with endometriosis [ß:- 0.003; 95%CI:- 0.468-0.461; p = 0.988)]. These results do not support data from the literature indicating a reduced aromatase expression in granulosa cells of affected women, but they highlight a potential subtle mechanism affecting the ovulation process in these women.

Transcriptomic signature of luteinized cumulus cells of oocytes developing to live birth after women received intracytoplasmic sperm injection.

OBJECTIVE: To compare the transcriptome of human cumulus cells (CCs) from oocytes with different outcomes (pregnancy yes/no, live birth [LB] yes/no), to identify noninvasive biomarkers for oocyte selection as well as new therapeutic targets to increase LB rates from assisted reproductive technologies (ART). DESIGN: Retrospective observational study. SETTINGS: This study was conducted at a University Hospital in Switzerland. PATIENTS: Subfertile couples undergoing controlled ovarian superstimulation and intracytoplasmic sperm injection with subsequent unbiopsied embryo transfer below the female age of 43 years. INTERVENTION(S): RNA sequencing of CCs from oocytes results in a pregnancy, no pregnancy, LB, or no LB. MAIN OUTCOME MEASURES: Differential gene expression (DEG) between CCs of oocytes results in "no pregnancy" vs. "pregnancy" and "pregnancy only" vs. "live birth." RESULTS: Although RNA sequencing did not reveal DEGs when comparing the transcriptomic profiles of the groups "no pregnancy" with "pregnancy," we identified 139 DEGs by comparing "pregnancy only" with "live birth," of which 28 belonged to clusters relevant to successful ART outcomes (i.e., CTGF, SERPINE2, PCK1, HHIP, HS3ST, and BIRC5). A functional enrichment analysis revealed that the transcriptome of CCs associated with LB depicts pathways of extracellular matrix, inflammatory cascades leading to ovulation, cell patterning, proliferation, and differentiation, and silencing pathways leading to apoptosis. CONCLUSION: We identified a CCs transcriptomic profile associated with LB after embryo transfer that, after further validation, could serve to predict successful ART outcomes. The definition of relevant pathways of CCs related to oocyte competency contributes to a broader understanding of the cumulus oocyte complex and helps identify further therapeutic targets for improving ART success.

Surveillance of clinical research integrity in medically assisted reproduction: a systematic review of retracted publications.

Background and purpose: Retraction is a significant consequence of scientific research, resulting from various factors ranging from unintentional errors to intentional misconduct. Previous reviews on retracted publications in obstetrics and gynecology have identified "article duplication," "plagiarism," and "fabricated results" as the main reasons for retraction. However, the extent of retracted articles in the literature on medically assisted reproduction (MAR) remains unclear. This systematic review aimed to assess the number and characteristics of retracted articles in the field of MAR. Methods: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for this study. A comprehensive literature search was conducted on the PubMed database from 1993 to February 2023, limited to English articles and including all 283 terms from the International Glossary on Infertility and Fertility Care. To identify retracted studies, a specific query combining the 283 terms from the glossary with a retraction-related keyword was used. Only studies focused on MAR and involving human subjects were included. Results: The electronic search yielded a total of 523,067 records in the field of infertility and fertility care. Among these, a total of 2,458 records were identified as retracted. The citation retraction rate was found to be 0.47% (2,458/523,067; 95%CI 0.45-0.49), and the citation retraction rate for randomized controlled trials (RCTs) was 0.20% (93/45,616; 95%CI 0.16-0.25). A total of 39 retracted articles specifically related to MAR were identified. Among these, 41.0% were RCTs (n = 16), 15.4% were reviews (n = 6), and 10.3% were retrospective studies (n = 4) or prospective studies (n = 4). Most of the retractions occurred shortly after publication, with "plagiarism" being the most common reason for retraction, followed by "duplicate publication." Discussion: The issue of retraction exists within the field of infertility and fertility care, including MAR. Our findings indicate that scientific misconduct, particularly plagiarism and duplicate publication, are the primary causes of retraction in MAR. Despite finding that the proportion of retracted citations is low, promoting scientific integrity should be a priority. The consequences of article retractions have significant implications for patient care and the scientific community. Hence, it is crucial to prioritize thorough screening of manuscripts before publication to maintain research integrity. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=185769, PROSPERO, identifier: CRD42020185769.

The impact of zygote vitrification timing on pregnancy rate in frozen-thawed IVF/ICSI cycles.

Introduction: Cryopreservation of bipronuclear (2PN) stage zygotes is an integral part of IVF laboratory practice in countries with strict embryo culture legislation. Vitrification of zygotes is compatible with several strategies in infertility treatments holding a freeze-all indication and allows for effective workload management in settings with limited resources. Although it yields high survival rates and clinical outcomes, the ideal timing to commence vitrification of zygotes is elusive while it is empirically practiced in the window between 17 and 21 h post-insemination (hpi). We aimed to deduce the association between pregnancy rate and the time interval from insemination (IVF and ICSI) to vitrification to contribute to the standardization ofzygote cryopreservation. Methods: A retrospective analysis of data on vitrification timings and pregnancy outcomes collected between 2011 and 2019 was performed. All included women received an embryo transfer after warming of vitrified zygotes at the 2PN stage. Results: A total of 468 embryo transfers were included of which 182 (38.9%) resulted in pregnancy and 286 (61.1%) not. Vitrification was on average performed 18.74 ±0.63 hpi in the pregnant group and 18.62 ± 0.64 hpi in the non-pregnant group (OR 1.36, 95% CI 1.01; 1.83, p = 0.045). A multivariate analysis controlling for uterine pathologies, maternal age, AMH, the number of MII oocytes, previous history of pregnancy success, endometriosis, AFC, nicotine intake and male factor infertility showed no predictive value of vitrification timing on pregnancy rate. Three time intervals between insemination and vitrification were defined: 17:00 to 18:00 hpi (Group A), 18:01 to 19:00 hpi (Group B) and 19:01 to 21:00 hpi (Group C). Pregnancy occurred in 40/130 women (30.80%) in Group A, in 115/281 women (40.90%) in Group B and in 27/57 women (47.40%) in Group C. Univariate but not multivariate analysis showed a significantly higher pregnancy rate after the latest time interval between insemination and 2PN vitrification when compared to the earliest (Group C vs. A, OR 2.03, 95% CI 1.07; 3.84, p = 0.031). Discussion: These findings encourage further investigation on the impact of vitrification timing on clinical outcomes and hold the potential to standardize cryopreservation of zygotes from IVF/ICSI cycles to eventually improve the quality of long-term ART outcomes.

Effects of vitamin D supplementation in endometriosis: a systematic review.

BACKGROUND: There is a growing body of human, animal and in vitro studies on vitamin D (vit D) substitution in endometriosis. The aim of this systematic review is to critically appraise and qualitatively synthesize the results of the available studies that examine the supplementation of vit D for endometriosis treatment. METHODS: A systematic search of the literature was conducted in four electronic databases (Medline, Cochrane, Scopus, Embase) and grey literature for original research articles on humans, animals and in vitro models published in any language. RESULTS: Four human studies, four animal studies and four in vitro studies were included. Quantitative synthesis of human studies showed no significant effect of vit D intake for dysmenorrhea (2 studies, 44 vit D vs 44 placebo, mean -0.71, 95% CI -1.94, 0.51) and non-cyclic pelvic pain (2 studies, 42 vit D vs 38 placebo, mean 0.34, 95% CI -0.02, 0.71). Regarding reproductive outcomes in women with endometriosis after in vitro fertilization, the only available study showed no differences between women taking vit D and women taking placebo. Three of the four included animal studies showed regression of endometriotic implants when treated with vit D. The in vitro studies demonstrated that vit D decreases invasion and proliferation of endometriotic lesions without affecting apoptosis. CONCLUSIONS: Although in vitro and animal studies suggest regression of the endometriotic implants and decrease of invasion and proliferation after vit D supplementation, this was not reflected in the results of the meta-analysis, which showed no benefit of vit D supplementation in patients with endometriosis and dysmenorrhea or non-cyclic pelvic pain as well as on the outcome of IVF treatment. However, given the heterogeneity and the diversity of the available studies, more research is required to shed light on the role of vit D supplementation in women with endometriosis.

Opportunities and Limits of Conventional IVF versus ICSI: It Is Time to Come off the Fence.

Conventional IVF (c-IVF) is one of the most practiced assisted reproductive technology (ART) approaches used worldwide. However, in the last years, the number of c-IVF procedures has dropped dramatically in favor of intracytoplasmic sperm injection (ICSI) in cases of non-male-related infertility. In this review, we have outlined advantages and disadvantages associated with c-IVF, highlighting the essential steps governing its success, its limitations, the methodology differences among laboratories and the technical progress. In addition, we have debated recent insights into fundamental questions, including indications regarding maternal age, decreased ovarian reserve, endometriosis, autoimmunity, single oocyte retrieval-cases as well as preimplantation genetic testing cycles. The "overuse" of ICSI procedures in several clinical situations of ART has been critically discussed. These insights will provide a framework for a better understanding of opportunities associated with human c-IVF and for best practice guidelines applicability in the reproductive medicine field.